Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
  • A61K2039/541—Mucosal route
  • A61K2039/543—Mucosal route intranasal
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55583—Polysaccharides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55588—Adjuvants of undefined constitution

Definitions

• Vacci ne composi tion s for intranasal admin stration compri sing chi tosan and use thereof
• This invention relates to vaccine compositions for intranasal administration, which compositions comprise one or more antigens and an effective adjuvant.
• the invention further relates to a method of immunising a mammal against diseases by administering such compositions to the mammal, methods of enhancing the immunogenicity of intranasally administered antigens, and uses of antigens in combination with an adjuvant for the manufacture of a vaccine composition for intranasal administration to immunise a mammal against specific diseases.
• Vaccines are preparations of antigenic materials, administered to recipients with a view to enhancing resistance to infection by inducing active immunity to specific microorganisms, for example bacteria or viruses.
• Vaccines which may be as single or mixed component vaccines, are presented in a variety of forms.
• current influenza vaccines consist of either inactivated whole virus, disrupted virus (split vaccines) or purified preparations of antigenic proteins.
• Vaccines are typically administered parenterally via injections.
• Traditional parenteral immunisation regimes are known to have a number of drawbacks. For example, many individuals possess a natural fear of injections and may experience psychological discomfort as a result. Furthermore, many individuals find injections physically uncomfortable.
• parenteral vaccination e.g. intramuscular, sub-cutaneous etc. is not an effective means of eliciting local antibody production if there has been no previous local exposure (e.g. by way of infection).
• the vaccine In the case of some diseases, it would be advantageous to stimulate the mucosal immune system. In order to do this, the vaccine must be applied topically to a mucosal surface. Thus, in certain cases (e.g. in the case of infections of the upper respiratory tract), it would be beneficial to obtain more effective stimulation of the local mucosal immune system of the respiratory tract.
• adjuvants have been shown, when co-administered with vaccine antigens, to further boost the effectiveness of vaccine compositions by stimulating the immune response (see e.g. Hibberd et al, Ann. Intern. Med. , 110, 955 (1989)).
• adjuvants which have been shown to be effective include interferon alpha, Klebsiella pneumoniae, glycoprotein and interleukin-2.
• Chitosans are derivatives of chitin or poly-N-acetyl-D-glucosamine in which the greater proportion of the N-acetyl groups have been removed through hydrolysis.
• European Patent Application 460 020 discloses pharmaceutical formulations including chitosans as mucosal abso ⁇ tion enhancers. That the chitosan could provide an adjuvant effect when administered in a vaccine composition is neither disclosed nor suggested.
• a vaccine composition adapted for intranasal administration, which composition comprises antigen and an effective adjuvant amount of a chitosan
• compositions according to the invention (hereinafter referred to as "the compositions according to the invention”).
• effective adjuvant amount will be well understood by those skilled in the art, and includes an amount of a chitosan which is capable of stimulating the immune response to nasally administered antigens, i.e. an amount that increases the immune response of a nasally administered antigen composition, as measured in terms of the IgA levels in the nasal washings.
• effective increases in IgA levels include by more than 5 %, preferably by more than 25% , and in particular by more than 50% , as compared to the same antigen composition without any adjuvant.
• Preferred concentrations of the chitosan in the compositions according to the invention are in the range 0.02 to 10% , more preferably 0.1 to 5 % and particularly 0.25 to 2% .
• the invention further provides a method of enhancing a protective IgA mucosal immune response and an IgG systemic immune response by administering intranasally to a mammal a vaccine composition comprising an antigen and an effective adjuvant amount of a chitosan.
• the antigen may be provided as a sub-unit of a cell wall protein or polysaccharide, or as DNA which produces the antigen in the cells after introduction of the DNA (e.g. by transfection). Strictly speaking, the DNA is not itself an "antigen" but it encodes the antigen and is termed antigen herein.
• the antigen may further be provided in a purified or an unpurified form. However, we prefer the antigen to be provided in a purified form.
• the invention may be applied to antigens including proteins from pathogens, recombinant proteins, peptides, polysaccharides, glycoproteins, lipopolysaccharides and DNA molecules (polynucleotides).
• influenza virus antigens such as haemagglutinin and neuraminidase antigens
• Bordetella pertussis antigens such as pertussis toxin, filamentous haemagglutinin, pertactin
• human papilloma virus (HPV) antigens HBV
• HCV human papilloma virus
• HCV human papilloma virus
• HCV human papilloma virus
• HPV human papilloma virus
• HPV human papilloma virus
• Hcobacter pylori antigens Helicobacter pylori antigens
• rabies antigens rabies antigens
• meningococcal antigens such as capsular polysaccharides of serogroup A, B, C, Y and W-135)
• tetanus antigens such as tetanus toxoid
• Preferred antigens include Bordetella pertussis antigens, meningococcal antigens, tetanus antigens, diphtheria antigens, pneumococcal antigens, tuberculosis antigens and RSV antigens.
• the antigen is not an influenza virus antigen.
• the chitosan is water-soluble, and may advantageously be produced from chitin by deacetylation to a degree of greater than 40% , preferably between 50% and 90% , and more preferably between 70% and 95% , deacetylation.
• deacetylated chitosans which may be mentioned include the "Sea Cure + " chitosan glutamate available from Protan Biopolymer A/S, Dra men, Norway.
• the molecular weight of the chitosan may be between 10 kD and 500 kD, preferably between 50 kD and 300 kD and more preferably between 100 kD and 300 kD.
• compositions according to the invention may be used in the immunisation of a host against diseases, for example as described in the tests below.
• a method of immunising a host against infection by disease comprises administering intranasally to the host a vaccine composition comprising antigen together with an effective adjuvant amount of a chitosan as hereinbefore defined.
• a method of enhancing the immune response of an intranasally administered antigen comprises co-administration of said antigen and a chitosan as hereinbefore defined.
• compositions according to the invention can be formulated as liquids or dry powders, for administration as aerosols, drops or insufflations.
• compositions according to the invention are formulated as dry powders or in the form of microspheres.
• compositions for administration as nasal drops may contain one or more excipients of the type usually included in such compositions, for example preservatives, viscosity adjusting agents, tonicity adjusting agents, buffering agents and the like.
• excipients for example preservatives, viscosity adjusting agents, tonicity adjusting agents, buffering agents and the like.
• a solution for intranasal administration preferably has a pH in the range 5.5 to 6.5, most preferably approximately pH 6.
• the present invention also provides a means for dispensing the intranasal compositions of purified surface antigen and chitosan.
• a dispensing device may, for example, take the form of an aerosol delivery system, and may be arranged to dispense only a single dose, or a multiplicity of doses.
• the vaccine will be administered to the patient in an amount effective to stimulate a protective immune response in the patient.
• the vaccine may be administered to humans in one or more doses, each dose containing 1 -250 micrograms and more preferably 2-50 micrograms of protein or polysaccharide antigen prepared from each viral or bacterial strain.
• haemagglutinin and neuraminidase preparations are prepared from three virus strains, e.g. 2 x Influenza A and 1 x Influenza B
• a total dose of viral protein administered may be in the range 15-150 micrograms.
• a total dose of bacterial protein administered as FHA, pertussis toxin (toxoid) or pertactin, either individually or in combination may be in the range 5-150 micrograms.
• FIG 1 illustrates the serum IgG anti-haemagglutinin response in mice immunised with purified surface antigen of influenza (PSA). Each bar represents the geometric mean titre of four mice. The error bars represent 1 standard error of the mean. The cut-off value is 50 which is the lower limit of detection.
• PSA surface antigen of influenza
• FIG 2 illustrates the nasal IgA anti-haemagglutinin response in mice immunised with purified surface antigen (PSA).
• PSA purified surface antigen
• Figures 3a and 3b illustrate the determination of nasal and pulmonary anti-haemagglutinin secreting cells of mice immunised with purified surface antigen (PSA), using ELISPOT.
• Figure 3a uses a log scale whilst Figure 3b uses a linear scale.
• Figures 4a, 4b and 4c illustrate the a ⁇ -Bordetella pertussis filamentous haemagglutinin (anti-FHA) serum IgG response, the anti-FHA secretory IgA response in lung lavage and the anti-FHA secretory IgA response, respectively, in nasal wash in mice immunised with FHA.
• anti-FHA anti-FHA
• Figure 5a, 5b and 5c illustrate the anti-FHA and anti-Pertussis toxin (toxoid; anti-PT) serum IgG response, lung lavage secretory IgA response and the nasal wash secretory IgA response, respectively, in mice immunised with FHA and PT.
• Example 1
• chitosan glutamate a medium viscosity deacetylated chitin having approximately 11 % residual N-acetyl groups
• the grade of chitosan glutamate used was "Sea Cure + 210", available from Protan Biopolymer A/S, Drammen, Norway.
• Influenza purified surface antigen containing both Influenza A and Influenza B protein, commercially available from Evans Medical Limited, Speke, Merseyside, United Kingdom, under the Trade Mark “Fluvirin” , was made up in phosphate buffered saline to give a protein concentration of approximately lmg/ml.
• the PSA consists almost entirely of the spike protein haemagglutinin (HA), although it does contain some neuraminidase.
• a 1 : 1 mixture of the chitosan glutamate solution and the PSA solution was prepared to give an intranasal vaccine composition containing 0.5 % chitosan glutamate (11 % acetylated), 0.8% NaCl, 0.05 % PSA and phosphate buffer to give a solution pH of 6.
• a composition comprising the same concentration of PSA adsorbed on to the known adjuvant Alhydrogel (aluminium hydroxide) was prepared. The PSA was adsorbed on to the Alhydrogel overnight at 40 °C.
• Alhydrogel aluminium hydroxide
• Example 2A The four compositions prepared as described in Example 1 were administered to groups of twelve adult (6-8 weeks) female BALB/c mice as follows:
• Group 4 20 ⁇ l chitosan solution administered intranasally.
• Group 5 20 ⁇ l PSA (10 ⁇ l per nostril) administered daily for three days. (Groups of four mice employed for this study).
• the immunisation procedure was carried out three times at monthly intervals, with the exception of Group 5 where the mice were immunised with three successive daily doses.
• the immunisation and sampling regime is shown in Table 1.
• mice from each group were terminally bled by cardiac puncture, their heads were removed and their nasal passages lavaged with 1 ml PBS + 1 % bovine serum albumin.
• Group 5 contained four mice only so blood was obtained by tail puncture for the first two samples and nasal washes were only performed at the third sampling point.
• lymphocytes were isolated from the mucous membranes of the nasal cavity and the lungs and the local immune response analysed by ELISPOT.
• Chitosan enhanced the serum response of intranasally administered PSA; after the third vaccination the antibody response in mice that received PSA + chitosan was 360-fold greater than that of mice receiving PSA alone I/N.
• the magnitude of the serum response in the PSA - chitosan mice was very similar to that of SIC immunised mice; in fact there was no statistical difference in the GMT's of the two groups at any sampling point (Student's t-Test p > 0.01).
• mice were immunised three times on successive days with PSA alone administered intranasally to study whether this regime had advantages over the once monthly regime. Although all the mice in this group had detectable serum antibodies 21 days after the first dose and the GMT at this time point was greater than in mice that had received a single dose of PSA intranasally, the number of mice seropositive decreased during the course of the study although the GMT did not (in this group the same mice were sampled at each time point). At the final time point the GMT of the mice on the monthly regime was an order of magnitude greater than mice on the daily regime. Table 2
• PSA + Alhydrogel given subcutaneously was very poor at inducing a nasal IgA response which is consistent with our previous findings and those of others.
• PSA alone given intranasally was also a poor mucosal immunogen although it was slightly better than subcutaneous immunisation in terms of the number of animals responding.
• Adding chitosan greatly boosted the IgA response, although the response was low after the first dose, HA-specific IgA could be detected in three out of four mice. The IgA response was boosted greatly in these mice by the second immunisation. The final immunisation had little effect; in fact the mean specific IgA levels had decreased slightly.
• ASO Local anti-HA antibody secreting cell response
• Lymphocytes were isolated from the nasal mucosa and lung parenchyma of groups of four mice at the third sampling point. Lymphocytes from individual mice were pooled and assayed for cells secreting IgA, IgG and IgM anti-flu antibodies using ELISPOT. The results are shown in Figures 3a and 3b.
• Bordetella pertussis filamentous haemagglutinin obtained from the National Institute for Biological Sciences, South Mimms, Potters Bar, London, United Kingdom, was made up in phosphate buffered saline (PBS) to give a protein concentration of approximately 1 mg/ml.
• PBS phosphate buffered saline
• 3C A 1 1 mixture of the CSN solution and the FHA solution was prepared to give an intranasal vaccine containing 0.5% CSN, 0.5 mg/ml FHA, 0.8 % sodium chloride and phosphate buffer, pH 6.0.
• compositions prepared as described above were administered to groups of 13 adult male Balb/c mice as follows:
• Group 1 20 ⁇ l (10 ⁇ l per nostril) FHA solution administered intranasally.
• FHA dose 10 ⁇ g
• Group 2 20 ⁇ l (10 ⁇ l per nostril) FHA/CSN solution administered intranasally.
• Group 3 50 ⁇ l FHA/Alhydrogel mixture administered subcutaneously.
• mice from each group at sampling points 1 and 2 and five mice from each group at sampling point 3 were terminally bled by cardiac puncture. After collection of blood samples the animals were killed with an intravenous overdose of pentobarbitone sodium and the nasal passages and the lungs were respectively lavaged with 1 ml PBS containing 1 % bovine serum albumin. Table 4
• 3F Anti-FHA IgG antibodies in the serum samples and anti-FHA secretory IgA antibodies in the nasal wash and lung lavage samples were measured by Enzyme Linked Immunosorbant Assay (ELISA).
• ELISA Enzyme Linked Immunosorbant Assay
• Serum IgG (IgG Eq.units/ml ⁇ SD) Lung lavage IgA (IgA Eq.units ml ⁇ SD) Nasal wash IgA (IgA Eq.units ml ⁇ SD)
• a solution of 2% (20 mg/ml) chitosan glutamate (CSN) was prepared by dissolving 200 g chitosan in 10 ml water.
• Bordetella pertussis filamentous haemagglutinin obtained from CAMR, Salisbury, Wiltshire, United Kingdom, was concentrated using an Aquacide II column and made up in phosphate buffered saline (PBS) to give a protein concentration of 267 ⁇ g/ml.
• Pertussis toxin PT
• PT non-toxic mutant, obtained from IRIS, Siena, Italy was concentrated using an Aquacide II column and made up in 0.5 M sodium chloride to give a final concentration of 267 ⁇ g/ml. Equal volumes of these two antigen solutions were mixed to give a solution containing each antigen at a final protein concentration of 133 ⁇ g/ml.
• a control solution for intranasal vaccination containing the same concentration of FHA and PT but not CSN was prepared.
• a negative control solution for intranasal vaccination containing 0.5 % CSN but neither FHA nor PT was also prepared by diluting one part 2% CSN solution with three parts PBS containing 0.0014 M potassium chloride and 0.3185 M sodium chloride.
• a mixture containing approximately 10 ⁇ g/ml FHA and 10 ⁇ g/ml PT adsorbed onto the known adjuvant Alhydrogel (200 ⁇ g/ml) was prepared. The antigens were adsorbed on to Alhydrogel by stirring the mixture for 30 min at room temperature.
• compositions prepared as described above were administered to groups of 10 adult female Balb/c mice as follows:
• Group 1 20 ⁇ l (10 ⁇ l per nostril) FHA/PT/CSN solution administered intranasally.
• Group 2 20 ⁇ l (10 ⁇ l per nostril) PT/FHA solution administered intranasally.
• Group 3 20 ⁇ l (10 ⁇ l per nostril) CSN solution administered intranasally
• CSN dose 100 ⁇ g
• Group 4 200 ⁇ l FHA/PT/Alhydrogel mixture administered intraperitoneally.
• the immunisation procedure was carried out twice at a monthly interval.
• the immunisation and sampling regime is shown in Table 6.
• mice from each group at sampling points 1 and 2 were terminally bled by cardiac puncture. After collection of blood samples the animals were killed with an intravenous overdose of pentobarbitone sodium and the nasal passages and the lungs were respectively lavaged with 1 ml PBS containing 1 % bovine serum albumin. Table 6
• the intraperitoneally administered FHA/PT solution (Group 4) elicited high primary and secondary responses to FHA and PT (Tables 7, 8 and Figure 5A). However, the secondary responses to FHA and PT were approximately 30 and 10 fold higher than the primary responses respectively.
• the negative control group serum samples (Group 3) were found to be negative both for anti-FHA and anti-PT IgG antibodies (Tables 7, 8).
• the lung lavage and nasal wash samples from the chitosan control group (Group 3) were found to be negative both for anti-FHA and anti-PT IgA antibodies (Tables 7, 8).
• the intraperitoneally administered FHA/PT/Alhydrogel (Group 4) was also found to produce no mucosal response (IgA antibodies) to either antigen in the lung lavage or the nasal washes.

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• 1995
  • 1995-12-07 GB GBGB9525083.3A patent/GB9525083D0/en active Pending
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